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Breast Cancer Research : BCR May 2022CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell...
BACKGROUND
CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell subtypes likely to spread into the circulatory system.
METHODS
The MDA-MB-231 cell line was used to model and visualize CCR5 activation by stimulation with RANTES, in an effort to quantify CCR5 endocytosis from the cell surface to the perinuclear space. CCR5 expression was then examined in tumor-associated cells (TACs), consisting of circulating tumor cells and circulating stromal cells, isolated from the peripheral blood of 54 metastatic breast cancer (mBC) patients to evaluate these CCR5 pooling patterns as they relate to progression and survival over 2 years.
RESULTS
In MB231 experiments, it was observed that CCR5 formed ~ 1 micron clusters identified as "CCR5 pools" on the surface of the cell, which in the presence of RANTES were endocytosed and translocated to the cell cytoplasm. When TACs from patients were analyzed, CCR5 pools were observed on the cell surface and translocating to the nuclear area, with CCR5 also having a positive statistical correlation between increased numbers of TACs and increased CCR5 pools on the cells. Further, it was determined that patients with very high numbers of CCR5 (> 10 CCR5 pools), specifically in the circulating stromal cells, were associated with worse progression-free survival (hazard ratio = 4.5, p = 0.002) and worse overall survival (hazard ratio = 3.7, p = 0.014).
CONCLUSIONS
Using a liquid biopsy approach, we evaluated two populations of tumor-associated cells emanating from primary tumors, with data suggesting that upregulation of the motility chemokine CCR5 in TACs provides clinically relevant opportunities for treating and tracking drug targetable receptors in mBC.
Topics: Breast Neoplasms; Chemokine CCL5; Endocytosis; Female; Humans; Neoplastic Cells, Circulating; Receptors, CCR5
PubMed: 35606863
DOI: 10.1186/s13058-022-01528-w -
Journal of Virology May 2022This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this...
This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.
Topics: Animals; CRISPR-Cas Systems; Chlorocebus aethiops; Gene Knockout Techniques; HEK293 Cells; HeLa Cells; Herpesvirus 1, Human; Humans; Membrane Transport Proteins; Nuclear Envelope; Nuclear Proteins; Proteomics; Vero Cells; Viral Proteins; Virus Release
PubMed: 35475666
DOI: 10.1128/jvi.00306-22 -
Journal of Virology May 2022Herpesviruses assemble new viral particles in the nucleus. These nucleocapsids bud through the inner nuclear membrane to produce enveloped viral particles in the...
Herpesviruses assemble new viral particles in the nucleus. These nucleocapsids bud through the inner nuclear membrane to produce enveloped viral particles in the perinuclear space before fusing with the outer nuclear membrane to reach the cytoplasm. This unusual route is necessary since viral capsids are too large to pass through nuclear pores. However, the transient perinuclear nucleocapsids (250 nm in diameter) are also larger than the width of the perinuclear space (30 to 50 nm). Interestingly, linker of the nucleoskeleton and cytoskeleton (LINC) components SUN and KASH connect the inner and outer nuclear membranes and regulate their spacing. Previous work by others on the related pseudorabies virus and human cytomegalovirus showed that they functionally interact with SUN proteins. To clarify the role of SUN proteins, we explored their impact on herpes simplex virus 1 (HSV-1), another herpesvirus. Using dominant negative SUN mutants and RNA interference, we show that HSV-1 propagation is dependent on the LINC complex. In contrast to pseudorabies virus, SUN2 disruption by either approach led to increased HSV-1 extracellular viral yields. This SUN2 dependency may be linked to its greater impact on perinuclear spacing in infected cells compared to SUN1. Finally, the virus itself seems to modulate perinuclear spacing. The large size of herpesviruses prevents them from travelling across the nuclear pores, and they instead egress across the two nuclear membranes, generating short-lived enveloped perinuclear virions. This poses a challenge as the perinuclear space is smaller than the virions. This implies the separation (unzipping) of the two nuclear membranes to accommodate the viral particles. The LINC complex bridges the two nuclear membranes and is an important regulator of perinuclear spacing. Work by others hint at its functional implication during pseudorabies virus and cytomegalovirus propagation. The present study probes the importance for HSV-1 of the SUN proteins, the LINC components found in the inner nuclear membrane. Using dominant negative constructs and RNA interference (RNAi), the data reveal that SUN2 exhibits antiviral propriety toward HSV-1, as disrupting the protein leads to increased viral yields. This is in contrast with that reported for pseudorabies and suggests that differences among herpesviruses may, once again, prevail.
Topics: Animals; Cell Nucleus; Herpesvirus 1, Human; Herpesvirus 1, Suid; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nuclear Envelope; Nucleocapsid; Virion
PubMed: 35435724
DOI: 10.1128/jvi.00453-22 -
Molecular Biology of the Cell May 2022How nuclear pore complexes (NPCs) assemble in the intact nuclear envelope (NE) is only rudimentarily understood. Nucleoporins (Nups) accumulate at the inner nuclear...
How nuclear pore complexes (NPCs) assemble in the intact nuclear envelope (NE) is only rudimentarily understood. Nucleoporins (Nups) accumulate at the inner nuclear membrane (INM) and deform this membrane toward the outer nuclear membrane (ONM), and eventually INM and ONM fuse by an unclear mechanism. In budding yeast, the integral membrane protein Brl1 that transiently associates with NPC assembly intermediates is involved in INM/ONM fusion during NPC assembly but leaving the molecular mechanism open. AlphaFold predictions indicate that Brl1-like proteins carry as common motifs an α-helix with amphipathic features (AαH) and a disulfide-stabilized, anti-parallel helix bundle (DAH) in the perinuclear space. Mutants with defective AαH (, ) impair the essential function of . Overexpression of promotes the formation of INM and ONM enclosed petal-like structures that carry Nups at their base, suggesting that they are derived from an NPC assembly attempt with failed INM/ONM fusion. Accordingly, expression triggers mislocalization of Nup159 and Nup42 and to a lesser extent Nsp1, which localize on the cytoplasmic face of the NPC. The DAH also contributes to the function of Brl1, and AαH has functions independent of DAH. We propose that AαH and DAH in Brl1 promote INM/ONM fusion during NPC assembly.
Topics: Membrane Proteins; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex Proteins; Protein Conformation, alpha-Helical; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35293775
DOI: 10.1091/mbc.E21-12-0616 -
PloS One 2022Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with...
Eosinophilic Esophagitis (EoE) is an antigen-triggered inflammatory condition of the esophageal lining characterized by eosinophilic infiltration. EoE is associated with significant remodeling, and although this remodeling is reversed by current treatment regimens, symptoms of EoE and associated remodeling reappear upon cessation of therapies. We hypothesized that structural remodeling of cell-cell adhesion is a key factor in the pathogenesis of EoE and that epithelial to mesenchymal transition (EMT) was a viable molecular process to lead to this remodeling. Endoscopically obtained biopsy samples from 18 EoE and 18 control pediatric patients were evaluated by transmission electron microscopy to measure intercellular spaces (IS) between cells. Biopsy samples from all groups were analyzed for cellular levels of cell-cell adhesion proteins: E-cadherin, zonula occludens associated protein-1 (ZO-1), and N-cadherin. We also analyzed for cellular levels and localization two of transcription factors, Twist1 and β-catenin, that are associated with promoting EMT. The IS was significantly increased in the EoE group compared to the control. We observed a significant decrease in E-cadherin and ZO-1 levels and a concomitant increase in N-cadherin levels in EoE samples compared to control. Further, while there was no significant change in cellular levels of β-catenin, we observed an altered localization of the protein from the cell membrane in control tissue to a nuclear/perinuclear localization in EoE. We observed higher levels of the transcription factor Twist1 in the EoE group compared to normal which was localized mainly at the nucleus. Our results suggest that the integrity of normally sealed esophageal epithelia is compromised in the EoE patients compared to control subjects, and this is due to alterations in the expression of cell adhesion molecules at the esophageal epithelium. Our data also suggest that EMT, potentially regulated by transcription factors β-catenin and Twist1, may be responsible for the molecular alteration which leads to the remodeling of esophageal epithelia in EoE.
Topics: Cadherins; Child; Eosinophilic Esophagitis; Epithelial-Mesenchymal Transition; Humans; Nuclear Proteins; Twist-Related Protein 1; beta Catenin
PubMed: 35239721
DOI: 10.1371/journal.pone.0264622 -
Plants (Basel, Switzerland) Feb 2022The response of chloroplasts to adverse environmental cues, principally increases in light intensity, stimulates chloroplast-to-nucleus retrograde signalling, which... (Review)
Review
The response of chloroplasts to adverse environmental cues, principally increases in light intensity, stimulates chloroplast-to-nucleus retrograde signalling, which leads to the induction of immediate protective responses and longer-term acclimation. Hydrogen peroxide (HO), generated during photosynthesis, is proposed to both initiate and transduce a retrograde signal in response to photoinhibitory light intensities. Signalling specificity achieved by chloroplast-sourced HO for signal transduction may be dependent upon the oft-observed close association of a proportion of these organelles with the nucleus. In this review, we consider more precisely the nature of the close association between a chloroplast appressed to the nucleus and the requirement for HO to cross both the double membranes of the chloroplast and nuclear envelopes. Of particular relevance is that the endoplasmic reticulum (ER) has close physical contact with chloroplasts and is contiguous with the nuclear envelope. Therefore, the perinuclear space, which transducing HO molecules would have to cross, may have an oxidising environment the same as the ER lumen. Based on studies in animal cells, the ER lumen may be a significant source of HO in plant cells arising from the oxidative folding of proteins. If this is the case, then there is potential for the ER lumen/perinuclear space to be an important location to modify chloroplast-to-nucleus HO signal transduction and thereby introduce modulation of it by additional different environmental cues. These would include for example, heat stress and pathogen infection, which induce the unfolded protein response characterised by an increased HO level in the ER lumen.
PubMed: 35214888
DOI: 10.3390/plants11040552 -
Anatomical Record (Hoboken, N.J. : 2007) Nov 2022Mesenchymal reticular cells (MRCs) form a supporting system in the cortex of the bursal follicle. The stellate-shaped MRCs exhibit a low electron density, which is...
Mesenchymal reticular cells (MRCs) form a supporting system in the cortex of the bursal follicle. The stellate-shaped MRCs exhibit a low electron density, which is helpful for their identification. A remarkable feature of MRC is the formation of multiple blebs in the nuclear envelope. The large, irregularly shaped blebs-which are perinuclear spaces-may be detached from the nuclear membrane, creating a sac-like granular endoplasmic reticulum (GER). Inside the bleb, membrane-bound bodies originate from cytoplasmic impressions. The cytoplasm contains a few round mitochondria, in which the internal membranes form either ovoid vesicles or the entire internal structure is indistinct. These mitochondria may be associated with the blebs. The classical Golgi complex with cis and trans faces cannot be recognized, but the accumulation of very small vesicles occurs around two or three stacked flat cisterns. The MRC forms a continuous layer along the corticomedullary basal lamina (CMBL), and during cell migration between the cortex and medulla, it may contribute to the temporary closure of the gap in the CMBL. At the outer surface of the cortex, transitory cells between the MRC and fibrocytes of the interfollicular connective tissue are present, and both cells can produce GER by blebbing. This finding suggests that MRCs and fibrocytes may have a common origin. The other stromal cell is the macrophage (Ma), which may fuse together to form multinucleated giant cells. The definition of histological classification of the third type of stromal cell is questionable, but certain morphological features may be referred to as progenitors of MRCs.
Topics: Animals; Bursa of Fabricius; Chickens; Cytoplasm; Stromal Cells
PubMed: 35142074
DOI: 10.1002/ar.24893 -
Archives of Razi Institute Oct 2021The soft and delicate tissue of the brain, which is the center of our coordination, is protected by its surrounding layers. The disruption of these layers results in...
The soft and delicate tissue of the brain, which is the center of our coordination, is protected by its surrounding layers. The disruption of these layers results in complicated situations and serious health problems. The brain has three protective layers of bone or skull tissue, the blood tissue layer, and finally the meningeal layer. The layer of blood tissue contains the blood vessels that are located between the skull and the meningeal membranes. If germs or foreign matter enter the fluid through the blood vessels under any circumstances and cause infection, the bones that protect the meninges will break and cause tissue damage. The present study aimed to assess the histological and immunohistochemical characteristics of the brain of rats that underwent induced acute purulent pneumococcal meningitis after antibiotic therapy with Ceftriaxone. A number of 20 white adult male Wistar rats were assigned to three groups. The first group (n=5) regarded as the control were injected with a saline solution into the subarachnoid space in an equivalent amount. The second and third groups of rats (n=5 and 10, respectively) were infected with acute purulent meningitis by the injection of 10 μl of Streptococcus pneumoniae (S. pneumonia) suspension into the subarachnoid space of the brain using a 23-G needle. The various areas of the brains of rats after meningitis induced by S. pneumoniae were examined after the treatment with Ceftriaxone. The S. pneumoniae culture was injected into the subarachnoid space in the area of the rhomboid fossa. Treatment started 18 h after the injection. On day 10, a repeated puncture was performed with the analysis of cerebrospinal fluid in order to confirm the absence of meningitis; thereafter, the animals were taken out of the experiment. No signs of meningitis were found on histological examination. Mild perivascular and pericellular focal edema were revealed with signs of overload of the lymphatic system in the brain and focal ischemic changes in neurons. The investigation of expression with caspase-3 revealed a positive reaction of individual neurons. A positive reaction with antibodies to NeuN and Doublecortin was detected in most neurons; moreover, Glial fibrillary acidic protein (GFAP)-positive astrocytes and their processes were visualized in all layers of the brain substance. The reaction with neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), CD 31, and CD 34 was negative. Typical structure and pictures pointed to an intact brain and purulent meningitis in the first and second groups. The microscopic image and the changes revealed during immunohistochemistry by dual corticosteroid antibodies and neuronal nuclear protein were characterized by predominantly cytoplasmic and perinuclear reactions, respectively. Some neurons are positive for caspase-3 and are related to changes in the characteristic of premature aging.
Topics: Animals; Male; Rats; Anti-Bacterial Agents; Brain; Ceftriaxone; Meningitis, Pneumococcal; Rats, Wistar
PubMed: 35096336
DOI: 10.22092/ari.2021.355885.1733 -
Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus.The Journal of Cell Biology Feb 2022In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and...
In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.
Topics: Autophagy; Autophagy-Related Proteins; Chromosomes, Fungal; Nuclear Envelope; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35061008
DOI: 10.1083/jcb.202103178 -
Viruses Nov 2021Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by... (Review)
Review
Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.
Topics: Capsid; Cell Nucleus; Cytoplasm; Herpesviridae; Herpesviridae Infections; Humans; Membrane Fusion; Nuclear Envelope; Phosphorylation; Simplexvirus; Viral Envelope; Viral Fusion Proteins; Virion; trans-Golgi Network
PubMed: 34960625
DOI: 10.3390/v13122356